Systems and methods for producing biofuels and related materials

ABSTRACT

Clostridium phytofermentans  cells (American Type Culture Collection 700394 T ) and all other strains of the species can ferment materials such as biomass into useful products and coproducts, such as ethanol, hydrogen and organic acids. Compositions that include  Clostridium phytofermentans  are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority from U.S. ProvisionalPatent Application No. 60/762,813, filed on Jan. 27, 2006, the contentsof which is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No.DE-FG02-02ER15330, awarded by the United States Department of Energy(DOE). The Government thus has certain rights in the invention.

TECHNICAL FIELD

This invention relates to compositions, and to systems and methods forproducing bio fuels such as ethanol, and related materials.

BACKGROUND

There is an interest in developing methods of producing usable energyfrom renewable and sustainable biomass resources. Energy in the form ofcarbohydrates can be found in waste biomass, and in dedicated energycrops, such as grains (e.g., corn or wheat) or grasses (e.g.,switchgrass). Cellulosic and lignocellulosic materials, are produced,processed, and used in large quantities in a number of applications.

A current challenge is to develop viable and economical strategies forthe conversion of carbohydrates into usable energy forms. Strategies forderiving useful energy from carbohydrates include the production ofethanol (“cellulosic ethanol”) and other alcohols (e.g., butanol),conversion of carbohydrates into hydrogen, and direct conversion ofcarbohydrates into electrical energy through fuel cells. For example,biomass ethanol strategies are described by DiPardo, Journal of Outlookfor Biomass Ethanol Production and Demand (EIA Forecasts), 2002;Sheehan, Biotechnology Progress, 15:8179, 1999; Martin, Enzyme MicrobesTechnology, 31:274, 2002; Greer, BioCycle, 61-65, April 2005; Lynd,Microbiology and Molecular Biology Reviews, 66:3, 506-577, 2002; andLynd et al. in “Consolidated Bioprocessing of Cellulosic Biomass: AnUpdate,” Current Opinion in Biotechnology, 16:577-583, 2005.

SUMMARY

The invention is based, in part, on the discovery of new characteristicsof an anaerobic bacterium, Clostridium phytofermentans. For example, anisolated strain of Clostridium phytofermentans (ISDg^(T), American TypeCulture Collection 700394¹) has been deposited under conditions thatassure that access to the cultures will be available during the pendencyof the patent application to one determined by the Commissioner ofPatents and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and35 U.S.C. 122. The deposits are available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction. Further, the subject culture deposits will be stored and madeavailable to the public in accord with the provisions of the BudapestTreaty for the Deposit of Microorganisms, i.e., they will be stored withall the care necessary to keep them viable and uncontaminated for aperiod of at least five years after the most recent request for thefurnishing of a sample of the deposits, and in any case, for a period ofat least 30 (thirty) years after the date of deposit or for theenforceable life of any patent which may issue disclosing the culturesplus five years after the last request for a sample from the deposit.The depositor acknowledges the duty to replace the deposits should thedepository be unable to furnish a sample when requested, due to thecondition of the deposits. All restrictions on the availability to thepublic of the subject culture deposits will be irrevocably removed uponthe granting of a patent disclosing them.

We have found that Clostridium phytofermentans, such as strain ISDg^(T),alone or in combination with one or more other microbes (e.g., yeasts orother bacteria) can ferment a material that is or includes acarbohydrate, or a mixture of carbohydrates, into a combustible fuel,e.g., ethanol, propanol and/or hydrogen, on a large scale. For example,Clostridium phytofermentans can ferment waste biomass, such saw dust,wood flour, wood pulp, paper pulp, paper pulp waste steams, grasses(e.g., switchgrass), biomass plants and crops (e.g., Crambe), algae,rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corncobs, corn grain (corn grind), distillers grains, and distillerssolutes, into ethanol, propanol and hydrogen. In addition, other usefulorganic products can also be produced, such as organic acids (e.g.,formic acid, lactic acid and acetic acid), or their conjugate bases(e.g., formate, lactate or acetate).

In one aspect, the invention features methods of making a fuel or fuelsfrom one or more biomass materials providing a biomass material thatincludes a high molecular weight carbohydrate; hydrolyzing the biomassmaterial to provide a hydrolyzed biomass material; combining thehydrolyzed biomass material with Clostridium phytofermentans cells in amedium; and fermenting the hydrolyzed biomass material under conditionsand for a time sufficient to produce a fuel or a mixture of fuels, e.g.,ethanol, propanol, and/or hydrogen. In addition to fuels, other productsand/or coproducts can be produced (e.g., organic acids and/or theirconjugate bases). In some embodiments, a concentration of carbohydratesin the medium is greater than about 20 mM. In other embodiments, theconcentration is greater than about 1 mM, e.g., greater than 2, 3, 4, 5,6, 7, 8, 9, 10, 12, 14, 16, or greater than about 18 mM.

In another aspect, the invention features a fuel plant that includes ahydrolysis unit configured to hydrolyze a biomass material that includesa high molecular weight carbohydrate, and a fermentor configured tohouse a medium and contains Clostridium phytofermentans cells dispersedtherein.

In another aspect, the invention features methods of making a fuel orfuels that include combining Clostridium phytofermentans cells and alignocellulosic material (and/or other biomass material) in a medium,and fermenting the lignocellulosic material under conditions and for atime sufficient to produce a fuel or fuels, e.g., ethanol, propanoland/or hydrogen.

In another aspect, the invention features methods of making a fuel orfuels that include combining Clostridium phytofermentans cells and amaterial that includes a carbohydrate in a medium, and fermenting thematerial including the carbohydrate under conditions and for a timesufficient to produce a fuel. A concentration of the carbohydrate in themedium is greater than 20 mM, e.g., greater than 30 mM, 40 mM, 50 mM, 75mM, or even 100 mM or more.

In any of the methods described herein, Clostridium phytofermentanscells can be utilized alone or in combination with one or more othermicrobes (e.g., yeasts or other bacteria) to produce a fuel or anotheruseful product, such as organic acids or their conjugate bases, whichcan be isolated as salts (e.g., sodium or potassium salts). An exampleof another bacterium is any strain of Zymomonas mobilis.

In another aspect, the invention features methods of making a fuel orfuels from one or more biomass materials with Clostridiumphytofermentans alone or in coculture with one or more other microbes,such as a yeast strain or a strain of Zymomonas mobilis. In addition tomaking fuels, the coculture can be used to make any coproduct describedherein, such as an organic acid, or a conjugate base or salt thereof.

In another aspect, the invention features methods of employingClostridium phytofermentans to produce an organic acid, or a conjugatebase or salt thereof, from one or more biomass materials, such as any ofthose materials described herein. For example, the other useful productsor coproducts can be used as feedstocks for the chemical orpharmaceutical industries. Examples of acids (conjugate bases) that canbe produced include lactic acid (lactate) and acetic acid (acetate).

In another aspect, the invention features cocultures that includeClostridium phytofermentans and one or more other microbes, e.g., yeastsor other bacteria (e.g., Zymomonas mobilis).

In another aspect, the invention features compositions that includeClostridium phytofermentans and one or more other microbes, e.g., yeastsor other bacteria (e.g., Zymomonas mobilis). The composition can be,e.g., in the form of a solid mixture (e.g., a freeze-dried mixture), ora liquid dispersion of the microbes, e.g., a coculture.

In another aspect, the invention features methods of making a usefulproduct, such as a biofuel, that include selecting a biomass or amixture of biomass materials; combining the biomass with a medium thatincludes Clostridium phytofermentans; fermenting the biomass for a firstperiod of time to provide a second biomass material; combining thesecond biomass material (with or without the Clostridiumphytofermentans) with another microbe or a mixture of microbes differentfrom Clostridium phytofermentans; and then fermenting the second biomassfor a second period of time to produce a useful material, such as a fuelor an organic acid.

In another aspect, the invention features fermentors that include amedium that includes Clostridium phytofermentans dispersed therein.Along with Clostridium phytofermentans, the medium can include one ormore of any of the other microbes described herein.

In another aspect, the invention features fermentors that includeClostridium phytofermentans in coculture with one or more of any of theother microbes described herein.

In another aspect, the invention features fermentors that include amedium that includes Clostridium phytofermentans dispersed therein. Thefermentors are configured to continuously remove a fermentation product,such as ethanol. In some embodiments, the concentration of the productremains substantially constant, or within about twenty five percent ofan average concentration. In some embodiments, any biomass describedherein is continuously fed to the fermentor.

In another aspect, the invention features products made by any of theprocesses described herein.

In another aspect, the invention features kits, e.g., for seeding afermentor, that include Clostridium phytofermentans. The kits canfurther include any one or more of any of the other microbes describedherein. For example, the microbes in the kits can be combined in asingle container or multiple containers. The microbes in the kits can bedispersed in a medium, or they can be freeze-dried. The kits can furtherinclude starter materials, such as nutrients.

Clostridium phytofermentans (American Type Culture Collection700394^(T)) is defined based on the phenotypic and genotypiccharacteristics of a cultured strain, ISDg^(T) (Warnick et al.,International Journal of Systematic and Evolutionary Microbiology,52:1155-60, 2002). The invention generally relates to systems, andmethods and compositions for producing fuels and/or other useful organicproducts involving strain ISDg^(T) and/or any other strain of thespecies Clostridium phytofermentans, which may be derived from strainISDg^(T) or separately isolated. The species is defined using standardtaxonomic considerations (Stackebrandt and Goebel, International Journalof Systematic Bacteriology, 44:846-9, 1994): Strains with 16S rRNAsequence homology values of 97% and higher as compared to the typestrain (ISDg^(T)) are considered strains of Clostridium phytofermentans,unless they are shown to have DNA re-association values of less than70%. Considerable evidence exists to indicate that microbes which have70% or greater DNA re-association values also have at least 96% DNAsequence identity and share phenotypic traits defining a species.Analyses of the genome sequence of Clostridium phytofermentans strainISDg^(T) indicate the presence of large numbers of genes and geneticloci that are likely to be involved in mechanisms and pathways for plantpolysaccharide fermentation, giving rise to the unusual fermentationproperties of this microbe. Based on the above-mentioned taxonomicconsiderations, all strains of the species Clostridium phytofermentanswould also possess all, or nearly all, of these fermentation properties.Clostridium phytofermentans strains can be natural isolates, orgenetically modified strains.

Advantages of the new systems and methods include any one of, orcombinations of, the following. Clostridium phytofermentans can fermenta broad spectrum of materials into fuels with high efficiency.Advantageously, waste products, e.g., lactose, waste paper, leaves,grass clippings, and/or sawdust, can be used to make fuels. Clostridiumphytofermentans remains active even at high concentrations ofcarbohydrates. Often materials that include carbohydrates can be usedraw, without pretreatment. For example, in some instances, it is notnecessary to pretreat the cellulosic material with an acid, a base, oran enzyme to release the lower molecular weight sugars that form part ofthe cellulosic material prior to fermentation. Instead, Clostridiumphytofermentans can ferment the raw cellulosic material into a fueldirectly. In some instances, lignocellulosic materials, e.g., sawdust orswitchgrass, can be used without removal of lignin, and/orhemicelluloses. Clostridium phytofermentans cells grow and ferment undera wide range of temperatures and pH ranges. The pH of the fermentationmedium may not need to be adjusted during fermentation. In someinstances, Clostridium phytofermentans cells can be used in combinationwith one or more other microbes to increase the yield of a desiredproduct, e.g., ethanol. In addition, Clostridium phytofermentans canferment high concentrations of 5-carbon sugars, or polymers that include5-carbon sugar repeat units, to combustible fuels. Five-carbon sugars,such as xylose, or polymers that include 5-carbon sugar repeat units,such as xylan and other components of the “hemicellulose” fraction ofplant cell walls, are hydrolyzed and fermented by Clostridiumphytofermentans concomitantly with other polymeric components oflignocellulosic materials yielding products such as ethanol andhydrogen. The 5-carbon sugars, or polymers that include 5-carbon sugarrepeat units, do not appear to divert metabolic resources of Clostridiumphytofermentans. Furthermore, Clostridium phytofermentans fermentshigher cellulose concentrations, e.g., greater than 40 mM (glucoseequivalents), with increasing ethanol yield. Other cellulose-fermentingmicrobes generally do not ferment higher concentrations of cellulose,above about 20 mM (glucose equivalents), and ethanol productiondecreases at higher cellulose concentrations (Desvaux et al., Appl.Environ. Microbiology, 66, 2461-2470, 2000).

Carbohydrates can be polymeric, oligomeric, dimeric, trimeric, ormonomeric. When the carbohydrates are formed from more than a singlerepeat unit, each repeat unit can be the same or different. Examples ofpolymeric carbohydrates include cellulose, xylan, pectin, and starch,while cellobiose and lactose are examples of dimeric carbohydrates.Example of a monomeric carbohydrates include glucose and xylose. Theterm “low molecular weight carbohydrate” as used herein is anycarbohydrate with a formula weight, or a number average molecular weightof less than about 1,000, as determined using a universal calibrationcurve. Generally, the term “high molecular weight carbohydrate” is anycarbohydrate having a molecular weight of greater than 1,000, e.g.,greater than 5,000, greater than 10,000, greater than 25,000, greaterthan 50,000, greater than 100,000, greater than 150,000, or greater than250,000.

For carbohydrates having a defined single structure with a defined andcomputable formula weight, e.g., monomeric, or dimeric carbohydrates(e.g., arabinose and cellobiose, respectively), concentrations arecalculated using the formula weight of the carbohydrate. Forcarbohydrates not having a defined single structure, e.g., polymericcarbohydrates (e.g., cellulose), concentrations are calculated assumingthat the entire mass of the polymeric carbohydrate can be hydrolyzed tothe monomeric carbohydrate unit from which the polymeric carbohydrate isformed. The formula weight of the monomeric carbohydrate unit is thenapplied to calculate the concentration in monomer equivalent units. Forexample, pure cellulose is made up entirely of glucose repeat units. 10grams of cellulose would give 10 grams of glucose, assuming that theentire mass of the cellulose is hydrolyzed to glucose. Glucose (C₆H₁₂O₆)has a formula weight of 180.16 amu. 10 grams of glucose is 0.056 molesof glucose. If this amount of glucose is in 1 L of solution, theconcentration would be 0.056 M or 56 mM. If the polymer has more thanone repeat unit, the concentration would be calculated as a totalaverage carbohydrate concentration by assuming that the entire mass ofthe polymeric carbohydrate can be hydrolyzed to the monomericcarbohydrate units from which the polymeric carbohydrate is formed. Forexample, if the polymeric carbohydrate is made up entirely of the tworepeat units, hydrolysis of X grams of polymeric carbohydrate gives Xgrams of monomeric carbohydrates. A composite formula weight is the sumof the product of the mole fraction of the first monomeric carbohydrateand its formula weight and the product of the mole fraction of thesecond monomeric carbohydrate and its formula weight. The average numberof moles of carbohydrates is then X grams divided by the compositeformula weight. The average carbohydrate concentration is found bydividing the average number of moles by the quantity of solution inwhich they reside.

A “fermentable material” is one that Clostridium phytofermentans (e.g.,ISDg^(T)) can, at least in part, convert into a fuel, e.g., ethanol,propanol or hydrogen and/or another useful product, e.g., an organicacid.

Biomass is an organic, non-fossilized material that is, or is derivedfrom, biological organisms (e.g., plants or animals), dead or alive.Biomass excludes mass that has been transformed by geological processesinto substances such as coal or petroleum, but includes materials thatare derived from living or dead organisms, e.g., by chemically treatingsuch organisms or remnants of such organisms. Examples of biomassinclude wood, wood-related materials (e.g., particle board), paper,grasses (e.g., switchgrass, Miscanthus), rice hulls, bagasse, cotton,jute, hemp, flax, bamboo, sisal, abaca, straw, leaves, grass clippings,corn stover, corn cobs, distillers grains, legume plants, sorghum, andbiomass crops (e.g., Crambe).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic cross-sectional view of a fermentation vesselholding a medium having Clostridium phytofermentans cells dispersedtherein.

FIG. 2 is a schematic cross-sectional view of a rotary knife cutter usedto fibrillate biomass.

FIG. 3A is a photograph of cellulosic material sheared in the rotaryknife cutter of FIG. 2.

FIG. 3B is highly enlarged photomicrograph of the material shown in FIG.3A.

FIG. 4 is a block diagram that shows a process for producing ethanol andhydrogen from biomass using acid hydrolysis pretreatment.

FIG. 5A is a block diagram that shows a process for producing ethanoland hydrogen from biomass using enzymatic hydrolysis pretreatment.

FIG. 5B is a block diagram that shows a process for producing ethanoland hydrogen from biomass using biomass that has not been enzymaticallypretreated.

FIG. 5C is a block diagram that shows a process for producing ethanoland hydrogen from biomass using biomass that has not been chemically orenzymatically pretreated, but is optionally steam treated.

FIG. 6 is a bar chart that shows major products and concentrations ofthe products obtained from fermenting various initial celluloseconcentrations (in glucose equivalents) together with Clostridiumphytofermentans.

DETAILED DESCRIPTION

FIG. 1 shows a fermentation vessel 10 that holds a medium 12 having afermentable material dissolved or dispersed therein. The fermentablematerial is or includes a carbohydrate, e.g., glucose, cellobiose, orcellulose. The medium 12 also has a plurality of Clostridiumphytofermentans cells 14 dispersed therein, such as ISDg^(T) cells. TheClostridium phytofermentans cells 14 ferment the fermentable material toproduce combustible fuel, e.g., ethanol and/or hydrogen. Other usefulproducts and coproducts can also be produced. Other products can includeorganic acids (e.g., formic acid, lactic acid and acetic acid), or theirconjugate base (e.g., formate, lactate or acetate ions).

Clostridium phytofermentans cells 14 (American Type Culture Collection700394^(T)) were isolated from damp silt in the bed of an intermittentstream in a forested site near Quabbin Reservoir in the state ofMassachusetts (USA). Generally, ISDg^(T) cells 14 are long, thin,straight, and motile rods (0.5 to 0.8 by 3.0 to 15.0 μm) that formround, terminal spores (0.9 to 1.5 μm in diameter). Additionalcharacteristics of Clostridium phytofermentans cells are described inWarnick et al., Int. J. Systematic and Evol. Microbiology, 52, 1155-1160(2002).

Clostridium phytofermentans cells 14 are cultured in an anaerobicenvironment, which is achieved and/or maintained by bubbling asubstantially oxygen-free gas through a bubbler 16 that includes gasoutlets 18 that are submerged below a surface 19 of the medium 12.Excess gas and effluent from reactions in the medium 12 fill headspace22, and are eventually vented through a gas outlet aperture 21 formed invessel wall 30. Gases that can be used to maintain anaerobic conditionsinclude N₂, N₂/CO₂ (80:20), N₂/CO₂/H₂ (83:10:7), and Nobel gases, e.g.,helium and argon. In some implementations, to achieve and/or maintainhomogeneity, medium 12 is stirred (as indicated by arrow 40).Homogeneity can also be maintained by shaking or vibrating vessel 10.

In some instances, the concentration of Clostridium phytofermentanscells 14 suspended in the medium 12 is from about 10⁶ to about 10⁹cells/mL, e.g., from about 10⁷ to about 10⁸ cells/mL. In someimplementations, the concentration at the start of fermentation is about10⁷ cells/mL.

We have found that Clostridium phytofermentans cells 14 can ferment bothlow, e.g., 0.01 mM to about 5 mM, and high concentrations ofcarbohydrates, and are generally not inhibited in their action atrelatively high concentrations of carbohydrates, which would haveadverse effects on other organisms. For example, the concentration ofthe carbohydrate in the medium can be greater than 20 mM, e.g., greaterthan 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 75 mM, 100 mM, 150 mM, 200 mM,250 mM, 300 mM, or even greater than 500 mM or more. In any of theseembodiments, the concentration of the carbohydrate is generally lessthan 2,000 mM.

The fermentable material can be, or can include, one or more lowmolecular weight carbohydrates. The low molecular weight carbohydratecan be, e.g., a monosaccharide, a disaccharide, an oligiosaccharide, ormixtures of these. The monosaccharide can be, e.g., a triose, a tetrose,a pentose, a hexose, a heptose, a nonose, or mixtures of these. Forexample, the monosaccharide can be arabinose, glyceraldehyde,dihydroxyacetone, erythrose, ribose, ribulose, xylose, glucose,galactose, mannose, fucose, fructose, sedoheptulose, neuraminic acid, ormixtures of these. The disaccharide can be, e.g., sucrose, lactose,maltose, gentiobiose, or mixtures of these.

In some embodiments, the low molecular weight carbohydrate is generatedby breaking down a high molecular weight polysaccharides (e.g.,cellulose, xylan or other components of hemicellulose, pectin, and/orstarch). This technique can be advantageously and directly applied towaste streams, e.g., waste paper (e.g., waste newsprint and wastecartons). In some instances, the breaking down is done as a separateprocess, and then the low molecular weight carbohydrate utilized. Inother instances, the high molecular weight carbohydrate is addeddirectly to the medium, and is broken down into the low molecular weightcarbohydrate in-situ. In some implementations, this is done chemically,e.g., by oxidation, base hydrolysis, and/or acid hydrolysis. Chemicalhydrolysis has been described by Bjerre, Biotechnol. Bioeng., 49:568,1996, and Kim et al., Biotechnol. Prog., 18:489, 2002.

In some implementations, the low molecular weight carbohydrate isgenerated by breaking down a polysaccharide using an enzyme or enzymes,e.g., endoglucanases, exoglucanases or cellobiohydrolases (CBH). Theseenzymes can be added to the polysaccharide source as enzymepreparations, or they may be made in-situ by an organism, e.g.,Aspergillus niger BKMF 1305, and Trichoderma reesei RUT C30. Enzymaticbreakdown has been discussed by T. Juhasz, Food Tech. Biotechnol.(2003), 41, 49.

In a specific implementation, lactose is used as the carbohydrate.Lactose is produced in large quantities by the cheese industry. Forexample, it has been estimated by Elliott, Proceedings of the 38^(th)Annual Marschall Cheese Seminar (2001), that about 470 million pounds oflactose per year are produced by the U.S. cheese industry, and another726 million pounds are produced in Europe. Lactose may be used in afermentor, e.g., a seed fermentor that feeds a main fermentor, as agrowth substrate for Clostridium phytofermentans cells alone, or alongwith other growth substrates. Lactose may be added to fermentationvessels to augment fermentation of low molecular weight carbohydratesand/or speed the decomposition and fermentation of cellulose, or otherhigh molecular weight carbohydrates.

The fermentable material can also be, or can include one or more highmolecular weight carbohydrates. High molecular weight carbohydratesinclude, e.g., polygalacturonic acid, cellulose, microcrystallinecellulose, pectin, starch, xylan, other hemicellulosic polymers, ormixtures of these. Microcrystalline cellulose and modifiedmicrocrystalline celluloses are available commercially from FMCBiopolymer under the trade name AVICEL®.

The fermentable material can also be, or can include, one or morebiomass materials, e.g., cellulosic or lignocellulosic materials.Cellulosic materials are those materials that include cellulose, butsubstantially no lignin, e.g., less than 0.5 percent by weight. Thecellulosic materials can be natural, semi-synthetic, or fully synthetic.For example, cotton is a natural cellulosic material. Semi-syntheticcellulosic materials include, e.g., rayon (regenerated cellulose) andtextiles which include cotton fibers, e.g., obtained from virgin scraptextile materials (e.g., remnants), or post consumer waste, e.g., rags.Other semi-synthetic cellulosic materials include distillers grains(e.g., from the corn ethanol industry), paper and products such aspolycoated paper and Kraft paper. The paper or paper products can bevirgin materials, or they can be post-consumer waste materials.

Lignocellulosic materials include cellulose and a percentage of lignin,e.g., at least about 0.5 percent by weight to about 60 percent by weightor more lignin. Lignin can be thought of as a polyphenolic material.Some lignins can be represented by Structure (I) below:

Lignins can be highly branched, and can also be partially crosslinked.Lignins can have significant structural variation that depends, at leastin part, upon its source, e.g., whether it is derived from a softwood,or a hardwood.

Lignocellulosic materials include, e.g., papermaking sludge; wood, andwood-related materials, e.g., saw dust, particle board or leaves; andnatural fiber sources, e.g., trees such as poplar trees, grasses such asswitchgrass, leaves, grass clippings, rice hulls, bagasse, jute, hemp,flax, bamboo, sisal, abaca, straw, corn cobs, corn stover, wheat straw,rice hulls, and coconut hair.

In particular implementations, the lignocellulosic material is obtainedfrom trees, such as Coniferous trees, e.g., Eastern Hemlock (Tsugacanadensis), Maidenhair Tree (Ginkgo bilboa), Pencil Cedar (Juniperusvirgineana), Mountain Pine (Pinus mugo), Deodar (Cedrus deodara),Western Red Cedar (Thuja plicata), Common Yew (Taxus baccata), ColoradoSpruce (Picea pungens); or Deciduous trees, e.g., Mountain Ash (Sorbus),Gum (Eucalyptus gunnii), Birch (Betula platyphylla), or Norway Maple(Acer platanoides), can be utilized. Poplar, Beech, Sugar Maple and Oaktrees may also be utilized.

In some instances, Clostridium phytofermentans cells can fermentlignocellulosic materials directly without the need to remove lignin.

However, in certain embodiments, it is useful to remove at least some ofthe lignin from lignocellulosic materials before fermenting. Forexample, removal of the lignin from the lignocellulosic materials canmake the remaining cellulosic material more porous and higher in surfacearea, which can, e.g., increase the rate of fermentation and ethanolyield. The lignin can be removed from lignocellulosic materials, e.g.,by sulfite processes, alkaline processes, or by Kraft processes. Suchprocess and others are described in Meister, U.S. Pat. No. 5,138,007,and Knauf et al., International Sugar Journal, 106:1263, 147-150 (2004).The lignin content of switchgrass is about 17.6% (percent dry weight),which is about the same as corn stover. The lignin content of writingpaper ranges from about zero percent lignin to about 12 percent lignin.Some office papers have a lignin content that is in the range of about11-12 percent lignin. Mosier et al., Bioresource Technology 96:673,2005, discusses the lignin content of some materials, and also somepretreatment strategies for removing it. If lignin is removed, it can beused as an energy source in the processes, e.g., to heat a boiler byburning the lignin.

Cellulosic materials can be obtained from lignocellulosic materials bychemically treating the lignocellulosic material to solubilize thelignin to a degree that allows the cellulosic material to be separatedfor the lignin, e.g., in the form of fibers. When the lignocellulosicmaterial is from trees, the dissolved lignin generally constitutesbetween about 25 to 45% of the material.

Materials can be reduced in size, e.g., by shearing the material in arotary knife cutter, or by pulverizing the material in a ball mill. Whena rotary knife cutter is used to reduce the size of the material, e.g.,a cellulosic or lignocellulosic material, typically the resultingmaterial is fibrous in nature, having a substantial length-to-diameterratio, e.g., greater than 5/1, greater than 10/1, greater than 15/1,greater than 20/1, or even greater than 25/1. When a ball-mill is used,typically the resulting material is in the form of flour, typicallyhaving substantially spherical particles, e.g., having a diameter ofless than 5 microns, e.g., less than 4, less than 2.5, less than 1micron.

FIG. 2 shows a rotary knife cutter 100 that includes a hopper 101 thatcan be loaded with a cellulosic or lignocellulosic material 102, e.g.,in the form of chips. The cellulosic or lignocellulosic material isdrawn into a shearing zone 103, and is sheared between stationary blades104 and rotating blades 106. A screen 105 prevents the cellulosic orlignocellulosic material from leaving the shearing zone 103 until thematerial is sized small enough to pass through apertures defined in thescreen. Once the cellulosic or lignocellulosic material has passedthrough openings in the screen, it is captured in bin 110. To aid in thecollection of the sheared fibrous cellulosic or lignocellulosicmaterial, bin 110 can be maintained at a pressure below nominalatmospheric pressure. The fibrous cellulosic or lignocellulosic materialcollected in the bin has a relatively low bulk density, e.g., less than0.5 grams per cubic centimeter, e.g., less than 0.3 grams per cubiccentimeter, or even less than 0.2 grams per cubic centimeter, and has a“fluffy” appearance, as shown in FIGS. 3A and 3B.

In some implementations, it can be desirable to use a fibrous materialthat has a relatively high surface area and/or a relatively highporosity. For example, a desirable fibrous material can have a surfacearea of greater than 0.5 m²/g, e.g., greater than 1.0 m²/g, 1.5 m²/g,1.75 m²/g, 5 m²/g, or even greater than 10 m²/g, as measured using BETBrunauer Emmett Teller surface area measurements); and/or a porosity ofgreater than 70 percent, e.g., greater than 80 percent, 87.5 percent, 90percent, or even greater than 95 percent, as determined mercuryporosimetry. High surface areas and/or high porosities can increasehydrolysis rate and/or fermentation rate.

Blends of any of the above materials can be used, e.g., blends ofmaterials obtained from paper sources, and materials obtained fromcotton.

In some embodiments, fermentors that include a medium that includesClostridium phytofermentans dispersed therein are configured tocontinuously remove a fermentation product, such as ethanol. In someembodiments, the concentration of the desired product remainssubstantially constant, or within about twenty five percent of anaverage concentration, e.g., measured after 2, 3, 4, 5, 6, or 10 hoursof fermentation at an initial concentration of from about 10 mM to about25 mM. In some embodiments, any biomass material or mixture describedherein is continuously fed to the fermentors.

The medium for Clostridium phytofermentans can include additionalconstituents, such as buffers, e.g., NaHCO₃, NH₄Cl, NaH₂PO₄.H₂O, K₂HPO₄,and KH₂PO₄; electrolytes, e.g., KCl, and NaCl; growth factors;surfactants; and chelating agents. Growth factors include, e.g., biotin,folic acid, pyridoxine.HCl, riboflavin, urea, yeast extracts, thymine,tryptone, adenine, cytosine, guanosine, uracil, nicotinic acid,pantothenic acid, B12 (Cyanocobalamine), p-aminobenzoic acid, andthioctic acid. Minerals include, e.g., MgSO₄, MnSO₄.H₂O, FeSO₄.7H₂O,CaCl₂.2H₂O, CoCl₂.6H₂O, ZnCl₂, CuSO₄.5H₂O, AlK(SO₄)₂.12H₂O, H₃BO₃,Na₂MoO₄, NiCl₂.6H₂O, and NaWO₄.2H₂O. Chelating agents include, e.g.,nitrilotriacetic acid. Surfactants include, e.g., polyethylene glycol(PEG), polypropylene glycol (PPG), copolymers PEG and PPG andpolyvinylalcohol.

In some implementations, fermentation conditions include maintaining themedium at a temperature of less than about 45° C., e.g., less than about42° C. (e.g., between about 34° C. and 38° C., or about 37° C.). In anyof these implementations, generally, the medium is maintained at atemperature above about 5° C., e.g., above about 15° C.

In some implementations, fermentation conditions include maintaining themedium at a pH of below about 9.5, e.g., between about 6.0 and 9.0, orbetween about 8 and 8.5. Generally, during fermentation, the pH of themedium typically does not change by more than 1.5 pH units. For example,if the fermentation starts at a pH of about 7.5, it typically does notgo lower than pH 6.0 at the end of the fermentation, which is within thegrowth range of the cells.

Clostridium phytofermentans cells adapt to relatively highconcentrations of ethanol, e.g., 7 percent by weight or higher, e.g.,12.5 percent by weight. Clostridium phytofermentans cells can be grownin an ethanol rich environment prior to fermentation, e.g., 7 percentethanol, to adapt the cells to even higher concentrations of ethanol,e.g., 20 percent. In some embodiments, Clostridium phytofermentans isadapted in successively higher concentrations of ethanol, e.g., startingwith 2 percent ethanol, then 5 percent ethanol, and then 10 percentethanol.

Products in addition to or other than ethanol can be produced. Moregenerally, fermentation products include fuels, such as alcohols (e.g.,ethanol, n-propanol, isopropanol, n-butanol, or mixtures of these) andhydrogen. Other products include organic acids (e.g., formic acid,lactic acid, acetic acid or mixtures of these), or their conjugate bases(e.g., formate, lactate or acetate ions) or salts thereof.

Clostridium phytofermentans, such as strain ISDg^(T), can be used aloneor in combination with one or more other microbes, such as yeasts orfungi (e.g., Saccharomyces cerevisiae, Pichia stipitis; Trichodermaspecies, Aspergillus species) or other bacteria (e.g., Zymomonasmobilis, Klebsiella oxytoca, Escherichia coli, Clostridiumacetobutylicum, Clostridium beijerinckii, Clostridium papyrosolvens,Clostridium cellulolyticum, Clostridium josui, Clostridium termitidis,Clostridium cellulosi, Clostridium celerecrescens, Clostridium populeti,Clostridium cellulovorans). For example, when a cellulolytic clostridium(strain C7) was grown in coculture with Zymomonas mobilis in a mediumcontaining cellulose as the growth substrate, ethanol yields were2.5-fold higher than in cultures with the clostridium alone (Leschineand Canale-Parola, Current Microbiology, 11:129-136, 1984). Mixtures ofmicrobes can be provided as solid mixtures (e.g., freeze-driedmixtures), or as liquid dispersions of the microbes, and grown incoculture with Clostridium phytofermentans, or microbes may be addedsequentially to the culture medium, for example, by adding anothermicrobe before or after addition of Clostridium phytofermentans.

In addition, any of the biomass materials described herein or mixturesof any of the biomass materials described herein can be treated with oneor more microbes described herein in a sequential or concurrent manner.For example, the biomass (or biomass mixture) can be treatedconcurrently with a mixture of microbes, e.g., a coculture, or thebiomass (or biomass mixture) can be initially treated with a firstmicrobe or a first mixture of microbes (e.g., one or more yeasts, fungior other bacteria) and then the resulting biomass can be treated withone or more stains of Clostridium phytofermentans. In other embodiments,the biomass material (or biomass mixture) is initially treated with oneor more stains of Clostridium phytofermentans and then the resultingbiomass is treated with one or more other microbes (any one of ormixtures of microbes described herein).

Large Scale Ethanol Production from Biomass

Generally, there are two basic approaches to producing fuel gradeethanol from biomass on a large scale utilizing of Clostridiumphytofermentans cells. In the first method, one first hydrolyzes abiomass material that includes high molecular weight carbohydrates tolower molecular weight carbohydrates, and then ferments the lowermolecular weight carbohydrates utilizing of Clostridium phytofermentanscells to produce ethanol. In the second method, one ferments the biomassmaterial itself without chemical and/or enzymatic pretreatment. In thefirst method, hydrolysis can be accomplished using acids, e.g., Brönstedacids (e.g., sulfuric or hydrochloric acid), bases, e.g., sodiumhydroxide, hydrothermal processes, ammonia fiber explosion processes(“EFEX”), lime processes, enzymes, or combination of these. Hydrogen,and other products of the fermentation can be captured and purified ifdesired, or disposed of, e.g., by burning. For example, the hydrogen gascan be flared, or used as an energy source in the process, e.g., todrive a steam boiler, e.g., by burning. Hydrolysis and/or steamtreatment of the biomass can, e.g., increase porosity and/or surfacearea of the biomass, often leaving the cellulosic materials more exposedto Clostridium phytofermentans cells, which can increase fermentationrate and yield. Removal of lignin can, e.g., provide a combustible fuelfor driving a boiler, and can also, e.g., increase porosity and/orsurface area of the biomass, often increasing fermentation rate andyield. Generally, in any of the to below described embodiments, theinitial concentration of the carbohydrates in the medium is greater than20 mM, e.g., greater than 30 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM,or even greater than 500 mM.

Ethanol Production from Biomass Utilizing Acid Hydrolysis Pretreatment

FIG. 4 illustrates a process 158 for producing ethanol from biomass byfirst treating biomass (e.g., between about 10 and about 60 weightpercent) suspended in water with an acid in an acidification unit 160.The biomass can be, e.g., wood chips, sawdust, milled agriculturalresidues or biomass crops (e.g., corn stover or switchgrass),corn-refining residue, sheared paper products like those shown in FIGS.3A and 3B, or mixtures of these and other cellulosic and/orlignocellulosic materials. The biomass can be acidified by bubblinggaseous sulfur dioxide through the biomass that is suspended in thewater, or by adding a strong acid, e.g., sulfuric, hydrochloric, ornitric acid. During the acidification, the pH is maintained at belowabout 3, e.g., below about 2.5 or below about 1.5. In addition to theacid already in the acidification unit, optionally, a metal salt such asferrous sulfate, ferric sulfate, ferric chloride, aluminum sulfate,aluminum chloride, magnesium sulfate, or mixtures of these can be addedto aid in the hydrolysis of the biomass. The biomass is held in theacidification unit 160, e.g., between about 1 and 6 hours, at atemperature of, e.g., between about 40° C. and about 80° C.

After acidification in the acidification unit 160, the biomass isde-watered in de-watering unit 164, e.g., by squeezing or bycentrifugation, to remove much of the acidified water. If desired, theacidified water can be re-used in the acidification unit 160.

The acid-impregnated biomass is fed into a hydrolysis unit 166, e.g., bya gravity feeder or rotary valve feeder that, in some instances, doesnot substantially densify the biomass. Steam is injected into thehydrolysis unit 166 to directly contact and heat the biomass to thedesired temperature. The temperature of the steam is, e.g., betweenabout 130° C. and about 220° C., and steam injection is continued for atime, e.g., of between about 10 minutes and about 120 minutes. Thehydrolysate is then discharged into flash tank 170 operating at atemperature of, e.g., between about 100° C. and about 190° C., and isheld in the tank 170 for a period of time, e.g., between about 1 hourand about 6 hours, to further hydrolyze the biomass, e.g., into solubleoligosaccharides and monomeric sugars.

The hydrolysate is then fed into extractor 172, e.g., a countercurrentextractor, a screw-conveyor extractor, or a vacuum belt extractor. Inextractor 172, the hydrolysate is washed with hot water at a temperatureof, e.g., between about 40° C. to about 90° C. For example, thehydrolysate is washed with a quantity of water greater than its ownweight, e.g., greater than two times its own weight, e.g., three times,four times, eight times, or even greater than ten times its own weight.

Alkali, e.g., in the form of lime or ammonia, is added to the extract inthe pH adjustment and filtration unit 180 to adjust the pH of theextract to between about 7 and about 8. Any precipitates during theaddition of the alkali are removed and the filtrate is forwarded to afermentor 182, which holds a medium that has Clostridium phytofermentanscells dispersed therein. The initial concentration of the carbohydratesin the medium is between 20 mM and about 100 mM. The concentration ofClostridium phytofermentans cells suspended in the medium is, e.g., fromabout 10⁷ to about 10⁹ cells/mL. In one implementation, the medium(referred to as GS-2) contains (each expressed in g/L) yeast extract,6.0; urea, 2.1; K₂HPO₄, 2.9; KH₂PO₄, 1.5; MOPS; 10.0; trisodium citratedihydrate, 3.0; cysteine hydrochloride, 2.0. In other implementations,components may be added to, or substituted for the components in theGS-2 medium, including: Tryptone, 2.0; adenine, 0.02; cytosine, 0.05;guanosine, 0.02; thymine, 0.05; uracil, 0.04; and a quantity of avitamin solution, e.g., 10 g/mL, prepared as described in Wolin et al.,Bacteriology, 87:993, 1964. The extract from the pH and filtration unit180 is adjusted so that the initial concentration of carbohydrates inthe medium is, e.g., between about pH 7.0 and pH 7.5.

If desired, at the start of the fermentation, in addition to thehydrolysate, a low molecular weight carbohydrate, e.g., lactose, can beadded to an initial concentration of, e.g., between about 1.0 g/L and 5g/L. This can help rapidly increase the number of Clostridiumphytofermentans cells and build enzymes within the fermentor.Fermentation is allowed to proceed while bubbling nitrogen gas throughthe medium for a period of time, e.g., between about 8 hours and 72hours, while maintaining a temperature of, e.g., between about 15° C.and 40° C. Hydrogen gas produced during the fermentation is swept fromfermentor 182 by the nitrogen gas, and is either collected or flared.

The extracted solids from the extractor 172 are de-watered, and then fedto second acidification unit 190. The solids from the extractor aresoaked in an aqueous solution of an acid, and optionally, a metal salt.During the acidification, the pH is maintained at below about 3, e.g.,below about 2.5 or below about 1.5. The biomass is held in the secondacidification unit 190, e.g., between about 1 and 6 hours, at atemperature of, e.g., between about 40° C. and about 80° C.

After acidification in the acidification unit 190, the biomass isde-watered in de-watering unit 200, e.g., by squeezing or bycentrifugation, to remove much of the acidified water. If desired, theacidified water can be re-used in the acidification unit 160 and/oracidification unit 190.

The acid-impregnated biomass is fed into second hydrolysis unit 202.Steam is injected into the second hydrolysis unit 202 to directlycontact and heat the biomass to a desired temperature. The temperatureof the steam and time of treatment is generally the same as used in thefirst hydrolysis unit 166. The hydrolysate is then discharged into aflash tank 204 operating at a temperature of, e.g., between about 140°C. and about 190° C., and is held in the tank 204 for a period of time,e.g., between about 0.5 and about 12 hours to further hydrolyze thebiomass.

Alkali is added to the extract in the pH adjustment and filtration unit210 to adjust the pH of the extract to between about 7 and about 8. Anyprecipitates during the addition of the alkali are removed, and thefiltrate is combined with the contents of fermentor 182, and thenforwarded to fermentor 212. Fermentation is allowed to proceed whilebubbling nitrogen gas through the medium for a period of time, e.g.,between about 15 hours and 100 hours, while maintaining a temperatureof, e.g., between about 25° C. and 35° C. Hydrogen gas produced duringthe fermentation is swept from fermentor 212 by the nitrogen gas, and iseither collected or flared.

After fermentation, the entire contents of fermentor 212 is transferredto distillation unit 220, and 96 percent ethanol/4 percent water (byvolume) is distilled and collected. Fuel grade ethanol (99-100 percentethanol) can be obtained by azeotropic distillation of the 96 percentethanol, e.g., by the addition of benzene and then re-distilling themixture, or by passing the 96 percent ethanol through molecular sievesto remove the water.

Ethanol Production from Biomass Utilizing Enzyme Hydrolysis Pretreatment

FIG. 5A illustrates a process 228 for producing ethanol from biomass byfirst treating biomass (between 10 and 60 weight percent), e.g.,suspended in water, with an enzyme or mixture of enzymes, e.g.,endoglucanases, exoglucanases, cellobiohydrolases (CBH),beta-glucosidases, glycoside hydrolases, glycosyltransferases, lyases,and esterases active against components of hemicellulsoe, pectin andstarch, in a hydrolysis unit 230. During the hydrolysis, the pH ismaintained between about 6.0 and about 7.5 by adding sodium hydroxide.The biomass is held in the hydrolysis unit 230, e.g., between about 6and 120 hours, at a temperature of, e.g., between about 25° C. and about40° C., and under nitrogen.

After hydrolysis, alkali, e.g., in the form of lime or ammonia, and/oracid, e.g., in the form of an aqueous solution of sulfuric acid, isadded to the contents of the hydrolysis unit 230 via pH adjustment unit234 to adjust the pH of the contents to between about 7 and about 8.After the pH is adjusted, the entire contents of hydrolysis unit 230 aretransferred to fermentor 240, which holds a medium that has Clostridiumphytofermentans cells dispersed therein. The initial concentration ofthe carbohydrates in the medium is between 20 mM and about 100 mM. Theconcentration of Clostridium phytofermentans cells suspended in themedium is, e.g., from about 10⁷ to about 10⁹ cells/mL. In oneimplementation, the medium contains (each expressed in g/L) yeastextract, 6.0, urea, 2.1, K₂HPO₄, 2.9; KH₂PO₄, 1.5; MOPS; 10.0; trisodiumcitrate dihydrate, 3.0; cysteine hydrochloride. The effluent fromhydrolysis unit 230 is adjusted so that the initial concentration ofcarbohydrates in the medium is, e.g., between about 50 and 200 mM. Ifdesired, at the start of the fermentation, cellobiose can be added to aninitial concentration of, e.g., between about 1.0 g/L and 5 g/L, orlactose can be added to speed fermentation or hydrolysis. Fermentationis allowed to proceed while bubbling nitrogen gas through the medium fora period of time, e.g., between about 8 hours and 72 hours, whilemaintaining a temperature of, e.g., between about 15° C. and 40° C.Hydrogen gas produced during the fermentation is swept from fermentor240 by the nitrogen gas, and is either collected or flared.

After fermentation, the entire contents of the fermentor 240 aretransferred to distillation unit 242, and fuel grade ethanol can beobtained as discussed above.

Ethanol Production from Biomass without Acid or Enzyme Pretreatment

FIG. 5B illustrates a process 250 for producing ethanol from biomass byfirst charging a holding vessel 252 with biomass, e.g., between 10 and60 weight percent, suspended in water. The biomass may be allowed tosoak for a time, e.g., of between about 1 hour and 36 hours at atemperature of, e.g., between about 25° C. and about 90° C. if undernormal atmospheric pressure, or between about 100 to about 175 if underpressures higher than normal atmospheric pressure, e.g., between about1.5 atmospheres and about 10 atmosphere. Alkali, e.g., in the form oflime or ammonia, and/or acid, e.g., in the form of an aqueous solutionof sulfuric acid, is added to the contents of the holding vessel 252after the soaking time via pH adjustment unit 260 to adjust the pH ofthe contents to between about 7 and about 8. After the pH is adjusted,the entire contents of the holding vessel 252 are transferred tofermentor 262, which holds a medium that has Clostridium phytofermentanscells dispersed therein. The initial concentration of the carbohydratesin the medium is between 20 mM and about 100 mM. Fermentation occurs infermentation vessel 262 under conditions that have been described above.Fuel grade ethanol is distilled in distillation unit 270, also asdescribed above.

FIG. 5C illustrates a process 300 for producing ethanol from biomass.Biomass (with or without lignin removed), and, optionally, steam ischarged to a fermentor 302. If lignin is removed, it can be used in anyenergy intensive process such as energy to drive a distillation unit.Steam can be advantageous to sterilize the biomass, and also to loosenthe biomass and make it more reactive. The biomass is charged to thefermentor 302 and water is added (if necessary) so that, e.g., betweenabout 10 and 60 weight percent of the total mass is suspended biomass.The biomass may be allowed to soak for a time, e.g., between about 1hour and 36 hours, at a temperature of, e.g., between about 25° C. andabout 90° C. if under normal atmospheric pressure, or between about 100°C. to about 175° C. if under pressures higher than normal atmosphericpressure, e.g., between about 1.5 atmospheres and about 10 atmosphere.Alkali, e.g., in the form of lime or ammonia, and/or acid, e.g., in theform of an aqueous solution of sulfuric acid, is added to the contentsof the fermentor 302 after the soaking time via pH adjustment unit 306to adjust the pH of the contents to between about 7 and about 8.

Seed fermentor 304, which holds a medium that has Clostridiumphytofermentans cells dispersed therein, is used to grow the Clostridiumphytofermentans cells. The concentration of Clostridium phytofermentanscells suspended in the medium is, e.g., about 10⁷ at the start ofgrowth, and about 10⁸ cells/mL when the seed mixture is ready for use toferment carbohydrates. The initial concentration of the carbohydrates inthe medium is between 20 mM and about 100 mM. In one implementation, themedium contains (each expressed in g/L) yeast extract, 6.0, urea, 2.1,K₂HPO₄, 2.9; KH₂PO₄, 1.5; MOPS; 10.0; trisodium citrate dihydrate, 3.0;and cysteine hydrochloride. The entire contents of the seed fermentor304 is transferred to fermentor 302 held at about room temperature, andallowed to ferment under conditions that have been described above. Fuelgrade ethanol is distilled in distillation unit 270, also as describedabove.

Ethanol Production from Biomass Utilizing a Combination of AcidHydrolysis Pretreatment, and Enzyme Hydrolysis Pretreatment

Ethanol from biomass can also be produced using a combination of acidhydrolysis pretreatment and enzyme hydrolysis pretreatment. For example,an initial hydrolysis can take place using an acid, e.g., by treatmentof the biomass in an acidification unit, followed by steam injection (asshown in FIG. 4), and then a final hydrolysis can be applied to theinitially hydrolyzed biomass using enzyme hydrolysis (as shown in FIG.5A).

Any combination of the ethanol production methods and/or features can beutilized to make a hybrid production method. In any of the methodsdescribed herein, lignin can be removed before fermentation.Furthermore, products in addition to or other than ethanol can beproduced by any of the methods described herein. More generally,fermentation products include fuels, such as alcohols and hydrogen, andother products, such as organic acids. Clostridium phytofermentans, suchas strain ISDg^(T), can be used alone, or synergistically in combinationwith one or more of any of the other microbes (e.g., yeasts or otherbacteria) described herein.

EXAMPLES

The disclosure is further described in the following examples, which donot limit the scope of the invention described in the claims.

In one experiment, Clostridium phytofermentans was grown in culturetubes in GS-2 cellulose medium at an initial pH of 7.5 under anatmosphere of N₂. The initial Clostridium phytofermentans concentrationwas about 0.8-1.1×10⁷ cells/mL and the temperature of incubation was 30°C.

FIG. 6 shows the concentration of ethanol (E), acetate (A), formate (F),and lactate (L) upon completion of cellulose decomposition as a functionof initial cellulose concentration (in glucose equivalents). At aninitial cellulose concentration of 37 mM, the concentrations of lactate(L), acetate (A), and ethanol (E), were 4 mM, 20 mM, and 59 mM,respectively. Formate (F) was not detectable at this initialconcentration. At an initial cellulose concentration of 74 mM, theconcentrations of lactate (L), formate (F), acetate (A), and ethanol(E), were 7 mM, 10 mM, 20 mM, and 123 mM, respectively; and at aconcentration of 148 mM, the concentrations of lactate (L), formate (F),acetate (A), and ethanol (E), were 10 mM, 17 mM, 20 mM, and 160 mM,respectively. FIG. 6 shows that high concentrations of cellulose do notinhibit the action of Clostridium phytofermentans, since theconcentration of ethanol (E) increases with increasing initialconcentration of cellulose.

This result contrasts with the results obtained using othercellulose-fermenting microbes that do not ferment higher concentrationsof cellulose, e.g., above about 40 mM (in glucose equivalents), andproduce decreased amounts of ethanol at higher cellulose concentrations(see Desvaux et al., Appl. Environ. Microbiology, 66, 2461-2470, 2000).It is also notable that when using Clostridium phytofermentans theacetate levels do not significantly increase with increasing initialconcentration of cellulose, which can be advantageous because more ofthe cellulose goes into making the more economically valuable ethanol.Generally, other cellulolytic bacteria produce less ethanol than acetate(on a molar basis) and ethanol-to-acetate ratios decrease withincreasing initial cellulose concentrations (for example, see Desvaux etal. above).

In a second experiment, Clostridium phytofermentans was grown in culturetubes in GS-2 medium containing cellulose at 25 or 50 mM (glucoseequivalents), or xylan at 25 or 50 mM (xylose equivalents), or celluloseplus xylan, each at 25 or 50 mM, for a total carbohydrate concentrationof 50 or 100 mM (monosaccharide equivalents). The initial pH of mediawas 7.5 and the initial Clostridium phytofermentans concentration was0.8-1.1×10⁷ cells/mL Cultures were incubated under an atmosphere of N₂at 30° C. Carbohydrate degradation was monitored visually.

In cultures containing both carbohydrates, cellulose and xylan weredegraded simultaneously. The rate of decomposition of cellulose or xylanin cultures containing both carbohydrates was equal to or greater thanthe rate of decomposition in cultures containing a single carbohydrate.This experiment demonstrates that the fermentation of cellulose bycultures of Clostridium phytofermentans is not inhibited by xylan, afive-carbon sugar polymer, and an important component of hemicellulose.Furthermore, this experiment shows that cellulose and xylan arefermented simultaneously by cultures of Clostridium phytofermentans,which can be advantageous given that most natural sources of biomasscontain mixtures of carbohydrates, with cellulose as the most abundantcomponent and hemicelluloses, such as xylan, second in abundance only tocellulose. In contrast, it appears that other microbes cannot fermentthe 5-carbon sugars, or polymers that include 5-carbon sugar repeatunits. Also, with other microbes, the 5-carbon sugars, or polymersthereof, can actually interfere with metabolic processes of the microbesto reduce fermentation rate and yield of ethanol.

In a third experiment, Clostridium phytofermentans was grown in culturetubes in GS-2 medium containing starch (Difco soluble starch) at 10, 20,or 40 g/L. The initial pH of media was 7.5 and the initial Clostridiumphytofermentans concentration was 0.8-1.1×10⁷ cells/mL. Cultures wereincubated under an atmosphere of N₂ at 30° C. Starch fermentation wasindicated by gas production and an increase in culture turbidity. Uponcompletion of fermentation, the concentrations of fermentation productswere determined. At an initial starch concentration of 10 g/L, theconcentrations of lactate, formate, acetate, and ethanol, were 1 mM, 2mM, 4 mM, and 69 mM, respectively. At an initial starch concentration of20 g/L, the concentrations of lactate, formate, acetate, and ethanol,were 3 mM, 4 mM, 5 mM, and 127 mM, respectively. At an initial starchconcentration of 40 g/L, the concentrations of lactate, acetate, andethanol, were 11 mM, 4 mM, and 132 mM, respectively. Formate was notdetected in this later experiment. These experiments indicate thathigher concentrations of starch do not inhibit the action of Clostridiumphytofermentans, since the concentration of ethanol increases withincreasing initial concentration of starch, a result analogous to thatdescribed above where cells were cultured with increasing concentrationsof cellulose.

In a fourth experiment, Clostridium phytofermentans was grown in culturetubes in GS-2 medium containing ground corn at 27 g/L, or wet distillersgrains at 10.5 g/L, or shredded corn stover at 20 g/L, or shreddedswitch grass at 20 g/L. The initial pH of media was 7.5 and the initialClostridium phytofermentans concentration was 0.8-1.1×10⁷ cells/mL.Cultures were incubated under an atmosphere of N₂ at 30° C. Allsubstrates were fermented, as indicated by gas production, and theprimary fermentation product in all cultures was ethanol. Thisexperiment indicates that Clostridium phytofermentans ferments thesecellulosic feedstocks to ethanol without chemical pretreatment of thecellulosic feedstock and without the addition of cellulases or otherenzymes.

In a final example, analyses of the genome sequence of Clostridiumphytofermentans support the conclusion that this microbe possessesunusual fermentation properties, and is particularly well suited todecomposing multiple components of plant biomass and fermenting thesecomponents to ethanol. The genome of Clostridium phytofermentans hasbeen sequenced by the Joint Genome Institute of the US Department ofEnergy. A draft sequence assembly was first available Nov. 8, 2005 andwas released to the public May 20, 2006(http://genome.ornl.gov/microbial/cphy/). This draft assembly contained4.5 MB of nucleotide sequence partitioned into 169 contiguous regions,from which 3671 putative proteins were derived. In December 2006, thegaps in the sequence were closed and the finished sequence is expectedearly in 2007.

As an indication of the unusual fermentation properties of Clostridiumphytofermentans and its ability to decompose multiple components ofplant biomass, we examined the genome sequence for evidence ofcarbohydrate uptake mechanisms. The genome of Clostridiumphytofermentans contains over 100 ABC-type transport systems and 52 ofthese appear to be dedicated to transporting carbohydrates into cells.While some of these transport systems are specific for monosaccharideslike glucose, fucose, or xylose, others are undoubtedly involved in thetransport of disaccharides (e.g., cellobiose), trisaccharides, andtetrasaccharides. This exceptionally broad diversity of carbohydratetransport systems is unprecedented among microbes, and indicates thatClostridium phytofermentans is particularly well suited to decomposingcellulosic biomass.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of making a fuel from biomass material, the methodcomprising: providing a biomass material comprising a high molecularweight carbohydrate; hydrolyzing the biomass material to provide ahydrolyzed biomass material; combining the hydrolyzed biomass materialwith Clostridium phytofermentans cells in a medium, wherein aconcentration of carbohydrates in the medium is greater than 20 mM; andfermenting the hydrolyzed biomass material under conditions and for atime sufficient to produce a fuel.
 2. The method of claim 1, wherein thefuel comprises an alcohol selected from the group consisting of ethanol,n-propanol, isopropanol, n-butanol, and mixtures thereof.
 3. The methodof claim 1, wherein the biomass material is hydrolyzed by treatment withan acid.
 4. The method of claim 1, wherein the biomass material ishydrolyzed by treatment with an enzyme or a mixture of enzymes.
 5. Themethod of claim 1, wherein the biomass material is hydrolyzed bytreatment with an acid, followed by treatment with an enzyme.
 6. Themethod of claim 1, wherein the biomass material is reduced in size priorto hydrolyzing.
 7. The method of claim 1, wherein the hydrolysis stepincludes pretreatment with acid in an acidification unit, followed byde-watering.
 8. A fuel plant comprising: a hydrolysis unit configured tohydrolyze a biomass material comprising a high molecular weightcarbohydrate; and a fermentor configured to house a medium andcomprising Clostridium phytofermentans cells dispersed therein.
 9. Thefuel plant of claim 8, wherein the fermentor configured to house themedium and comprising Clostridium phytofermentans further comprises oneor more other microbes different than Clostridium phytofermentans. 10.The fuel plant of claim 9, wherein the one or more other microbescomprise one or more yeasts.
 11. The fuel plant of claim 9, wherein theone or more other microbes comprise one or more bacteria.
 12. The fuelplant of claim 11, wherein the one or more bacteria comprise any one ormore stains of Zymomonas mobilis.
 13. A method of making a fuel, themethod comprising: combining Clostridium phytofermentans cells and alignocellulosic material in a medium; and fermenting the lignocellulosicmaterial under conditions and for a time sufficient to produce a fuel.14. The method of claim 13, wherein the lignocellulosic material isselected from the group consisting of wood, wood pulp, papermakingsludge, paper pulp waste streams, particle board, grasses, rice hulls,bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corncobs, corn stover, distillers grains, leaves, wheat straw, coconut hair,algae, switchgrass, Miscanthus, legume plants, sorghum, biomass crops(Crambe), and mixtures thereof.
 15. The method of claim 13, wherein themedium has dissolved therein constituents selected from the groupconsisting of growth factors, minerals, surfactants, chelating agents,and mixtures thereof.
 16. The method of claim 13, wherein conditionsinclude maintaining the medium at a temperature of less than about 45°C.
 17. The method of claim 13, wherein conditions include maintaining apH of the medium below about 9.5.
 18. The method of claim 13, whereinthe fuel is ethanol, and wherein conditions include maintaining aconcentration of the ethanol lower than about 1 M.
 19. A method ofmaking a fuel, the method comprising: combining Clostridiumphytofermentans cells and a material comprising a carbohydrate in amedium, wherein a concentration of the carbohydrate in the medium isgreater than 40 mM; and fermenting the material comprising thecarbohydrate under conditions and for a time sufficient to produce afuel.
 20. The method of claim 19, wherein the concentration is greaterthan 50 mM.
 21. The method of claim 20, wherein the concentration isgreater than 100 mM.
 22. The method of claim 21, wherein theconcentration is less than 2000 mM.
 23. The method of claim 19, whereinthe material comprises a low molecular weight carbohydrate having amolecular weight of less than 1,000.
 24. The method of claim 23, whereinthe low molecular weight carbohydrate is selected from the groupconsisting of arabinose, cellobiose, fructose, galactose, glucose,lactose, mannose, ribose, xylose, and mixtures thereof.
 25. The methodof claim 19, wherein the material comprises a high molecular weightcarbohydrate having a molecular weight of greater than 1,000.
 26. Themethod of claim 25, wherein the high molecular weight carbohydrate isselected from the group consisting of cellulose, microcrystallinecellulose, polygalacturonic acid, pectin, starch, xylan, and mixturesthereof.
 27. The method of claim 19, wherein the material is pulverizedsuch that it has a particle size of between about 5 microns and 50microns.
 28. The method of claim 19, wherein the material comprises ablend of two or more carbohydrates.
 29. The method of claim 19, whereinthe material comprises a cellulosic or lignocellulosic material.
 30. Themethod of claim 29, wherein the cellulosic or lignocellulosic materialis selected from the group consisting of polycoated paper, Kraft paper,papermaking sludge, wood, wood pulp, distillers grains, particle board,leaves, grasses, grass clippings, rice hulls, bagasse, cotton, jute,hemp, flax, bamboo, sisal, abaca, straw, corn cobs, corn stover, wheatstraw, coconut hair, algae, switchgrass, crambe, biomass crops, andmixtures thereof.
 31. The method of claim 19, wherein the materialcomprises a low molecular weight carbohydrate formed by breaking down ahigh molecular weight carbohydrate.
 32. The method of claim 31, whereinthe breaking down includes using an enzyme.
 33. The method of claim 32,wherein the enzyme is selected from the group consisting ofendoglucanases, exoglucanases, cellobiohydrolases (CBH), β-glucosidase,glycoside hydrolases, glycosyltransferases, lyases, and esterases activeagainst components of hemicellulose, pectin, and starch, and mixturesthereof.
 34. The method of claim 19, wherein the material issubstantially free of lignin.
 35. A method comprising: combiningClostridium phytofermentans cells, a second microbe and a materialcomprising a carbohydrate in a medium; and fermenting the materialcomprising the carbohydrate.
 36. The method of claim 35, wherein thefermenting is carried out under conditions and for a time sufficient toproduce a fuel.
 37. The method of claim 35, wherein the second microbecomprises a yeast.
 38. The method of claim 35, wherein the secondmicrobe comprises a bacterium different than Clostridiumphytofermentans.
 39. A composition comprising Clostridiumphytofermentans and a second microbe.